The phenomenon of osteoinduction was recognized by Lancroix in 1945 when he demonstrated that acid alcohol bone extracts induced heterotopic bone formation in ectopic sites. Twenty years later Urist and his co-workers decalcified bone matrix and observed new cartilage and bone formation when implanted intramuscularly. These discoveries led to isolation and purification of bone inducing agent named BMPs from bone matrix of different species and years later to cloning and characterization of several cDNAs encoding these novel proteins. The biological activity of BMPs has been determined by bioassay in rat or mouse muscle bounces or by ALP measurements in mammalian cell cultures.
Previous studies since 1965 have shown that BMPs are part of the TGF-β superfamily and like all the family members they have multiple effects on cell migration, growth and differentiation especially in bone formation and tissue repair but also in embryogenesis or cancer. They are low molecular weight hydrophobic glycoproteins which are soluble to chaotrophic agents such as urea and guanidinium hydrochloride but are resistant to several proteases, for example collagenases.
BMPs are produced as large precursor molecules which are processed proteolytically to mature peptides after the translation. Like all the members of TGF-β superfamily, BMPs have the pattern of seven cysteine residues in their C-terminal mature region. Between these cysteines there are three disulfide bonds within mature BMP monomers and one disulfide bond which combines two monomers into a biologically active BMP dimer.
BMPs act through specific transmembrane receptors located on cell surface of the target cells. The BMP receptors are serin-threonin kinases which resemble TGF-β receptors and are divided into two subgroups: type I and type II receptors. BMPs can bind strongly only to the heterotetrameric complex of these receptors. This complex formation is essential to the BMP signal transduction. Inside the target cell, BMP signals are transmitted to the nucleus via specific signal molecules called Smads, which are also responsible for suppression of BMP signals.
Until now, 16 different BMPs have been characterized and seven of them (BMPs 2-7 and 9) have shown to be able to induce bone formation when implanted in ectopic sites. According to the amino acid sequence of the mature part these BMPs are divided into two subgroups. BMPs 2 and 4 are 86% identical and BMPs 5, 6 and 7 are 78% identical. Between these two groups the identity is only about 56%. The amino acid sequence of BMP-3 is about 45% alike with BMPs 2 and 4 and BMP-9 is 50-55% identical with BMPs 2, 4, 5, 6 and 7. Due to high homology and small variety in size, BMPs are quite difficult, very time consuming and expensive to separate, purify and identify from each other at protein level. This is the reason why most of the BMPs are nowadays being produced using molecular biological tools. Different kinds of recombinant protein techniques have been tested and both eukaryotic and prokaryotic systems have been utilized.
Majority of research has focused on human recombinant BMPs, but with regard to effective bone induction antlers of Cervidae family form an interesting research area. Antlers are bony cranial organs typical to the Cervidae family and they differ from Bovidae horns in their growing pattern. Antlers grow from the tip and males cast them away once per year. It has been suggested that antlers are the fastest growing structures through the mammalian species and they are known to be the only structures that regenerate completely every year. Antlers are formed by modified endochondral ossification meaning that the process is performed through the highly vascularized cartilage model which is calcified and finally transformed into bone. Antlers form an interesting model of adult regenerating mineralized tissue and bone remodeling has been shown to continue until the time of antler casting. Although the ultimate reason for the amazing speed of antler growth has not yet been resolved, antlers have been shown to contain several BMPs. Deer antler has been proven to express BMP-2 and BMP-4 (Feng et al. 1997 Biochim Biophys Acta 1350:47-52; Feng et al. 1995 Biochim Biophys Acta 1263:163-168). In addition reindeer antlers express BMP-3b (Kapanen et al. 2002 J Biomed Mat Res 59:78-83). Yet, it is also possible that there is one or more totally uncovered factor(s) which are responsible for antler growth speed.
Due to their osteoinductive capacity, both BMPs extracted from demineralized bone matrix and BMPs produced by recombinant technique are very interesting and highly potential alternatives to bone grafting. Different BMPs have been used in many experimental and clinical studies.
Bone morphogenetic protein 4 has been isolated from different origins including some mammalian species like human, mouse, rat, rabbit and dog. Like BMP-2 it has also been isolated from antler of Texas fallow deer a member of Cervidae family (Feng et al. 1997; Feng et al. 1995). However, the functionality and effect of deer BMP-4 has not been demonstrated as it has not been produced in any expression systems.
Until now, BMP-3b has been the only BMP characterized in reindeer antler tissue (Kapanen et al. 2002).
Several in vitro studies have shown that BMP-4 signaling is required for chondrogenesis to convert chondrogenic precursor cells into chondrocytes. Its expression is enhanced by the impact of fracture and like BMP-3, -7 and -8 it shows a restricted period of expression from day 14 to 21. It is expressed in callus-forming tissue before callus formation. It also acts as a chemoattractive protein for primary human mesenchymal progenitor cells. Furthermore, BMP-4 has been shown to play a critical role in induction of bone formation both in vivo and in vitro and like BMP-2 its influence on embryogenesis can not be replaced by any other BMP. Though, functions and activity of BMP-4 have been under constant study for over the decade and though human BMP-4 has been produced as recombinant protein, until now, nobody has published information of the biological activity of BMP-4 cloned from the antler tissue of any member of Cervidae family.
U.S. Pat. No. 6,245,889 discloses purified human BMP-2 and BMP-4 proteins and processes for producing them. Also a pharmaceutical composition comprising said BMP-4 is disclosed. As generally known in the art, these proteins and compositions may be used in the treatment of bone and cartilage defects and in wound healing and related tissue repair. Further, said pharmaceutical composition may include a matrix capable of delivering said BMP proteins to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.
U.S. Pat. No. 5,399,677 discloses DNA molecules encoding mutant forms of bone morphogenetic proteins. The mutant forms of BMP can be produced bacterially and refolded to produce biologically active homodimers or heterodimers of BMP. A method of making such mutant BMP is also disclosed. Said mutant forms are useful since they are correctly folded when produced in bacterial hosts.
WO 98/51354 discloses osteogenic devices and methods of use thereof for repair of bone and cartilage defects. The method for producing new bone growth at bone defect site in a mammal comprises the step of implanting in a defect site a calcium phosphate matrix comprising at least one osteogenic protein. Said osteogenic proteins include several morphogens, such as bone morphogenetic proteins.
EP 1131087 discloses further use for morphogenetic proteins, such as BMP proteins. It is shown that exposing cancer cells to morphogens inhibits cancer cell growth and causes such cells to differentiate away from the cancerous phenotype. The use of morphogen can influence cancer cell fate and, in turn, alleviate the symptoms of cancer. Preferred morphogens disclosed include BMP-4.
Although some applications of known BMP proteins as bone and cartilage forming inducers or for alleviating the symptoms of cancer are already known, there is still need for better methods for isolating such proteins and for better morphogenetic proteins, for example ones which possess more efficient bone forming properties or are more soluble. Such proteins would be useful for better therapeutic methods and applications. Also methods for producing such proteins would be useful.